| Peanut disease caused by Elsino? arachidis(Bitanc.& Jenkins)Rossman & W.C.Allen has been increasing year by year,and it has become one of the most serious diseases in peanut production in China,which seriously threatens the healthy and sustainable development of the peanut industry.E.arachidis is difficult to isolate,and it grows slowly under artificial culture conditions and is not easy to produce conidia.The subsequent research on the fungus ha s caused be hindered.Therefore,the biological characteristics,sporulation method and infection process of E.arachidis has been studied to lay the theoretical foundation for the prevention and control technology of peanut scab.1.Biological Characteristics of Elsino? arachidis causing peanut scabThe biological characteristics of E.arachidis were investigated to clarify the characteristics of the pathogen.The results showed that the colony morphology of E.arachidis on PDA appeared raised wrinkled and grew slowly.The colonies diameter of strains LN-SY-A01,LN-FT-A01 and LN-JB-C01 grown on PDA for 30 days were only 26.37 mm,26.43 mm and 27.07 mm,respectively.The optimum growth condition for the isolates LN-SY-A01 and LN-FT-A01 were PDA,PSA and OA with mannose(carbon source),yeast extract(nitrogen source),at 25℃ under p H4-6;The optimum growth condition for the isolate LN-JB-C01 were PDA and PSA with galactose(carbon source),yeast extract(nitrogen source),at 25℃ under p H4-6 in the dark.The best carbon and nitrogen sources were galactose and yeast extract.The mycelia could not survive above 48 ℃ for 10 min.The optimum temperature for conidial germination was 25℃,best p H value was 5.The best carbon and nitrogen sources were 1% dextrose,1% alanine and 1% glycine.Light had little effect on conidial germination.The conidia could not germinate above 47℃ for 10 min.2.Screening of sporulate method for E.arachidisIn order to induce the conidia of the fungus to be used for subsequent research,seven kinds of experiments for inducing sporulation of E.arachidis are designed.Screening efficient method to produce conidia.The results showed that the fungus could successfully produce conidia by mycelial coating method,Fries method and plug method in cereal medium.The use of pulg inoculation method for sporulation is the simplest,and the conidia vigor is the highest,but the conidia maturity is not uniform,the different stages of spores and sporulation structure can be observed at the same time,and sporogenous structure is occasionally observed.However,the conidia produced by this method are more adhesive,difficult to separate from the culture medium and hyphae,and the spore suspension cannot be obtained.This method can be used to identify pathogens.The conidia produced by the coating method are relatively uniform in size,uniform in maturity,and capable of obtaining the spore suspension,which is easy to count sporulation,and can be used for experiments such as quantitative inoculation.However,the amount of spores produced is small,and the number of a colony is only 2×104.Therefore,the sporulation method needs to be further optimized.The Fries method can produce a large number of conidia,but the success rate is extremely low and unstable,and whether sporulation and the number of conidia are accidental,the method often produces chlamydospores.Therefore,the specific sporulation conditions of this method are for further investigation.3.Optimization of sporulation conditions of E.arachidisIn order to carry out serious researches on pathogenicity,epidemiology and molecular genetics of the fungal which requires conidia.In this paper,a simple and efficient method for conidial production is established by optimized the coating method.The nutritional and environmental conditions required to sporulate were strict: the colonies should be inoculated for 5 to 7 days on PDA with p H 7 in continual darkness as a preculture,and ensuring the density was maintained at 5.0 colonies per cm2 or less.Then,grown colonies should be cultured in water for 14 h to produce sufficient conidia.The maximum sporulation of isolate LN-FT-A01 was 8×104.The 20 isolates tested could produce conidia by this method,and the sporulation ability between the strains was significantly different,and the sporulation amount ranged from 0.7×104 to 2.3×105.The conidia produced by this method can infect peanut leaves in vivo or vitro,causing the leaves to produce typical symptoms of peanut scab.With the increase of the content of elsinochrome(ESC)in PDA,the conidia production decreased,and the conidia were almost produced when the ESC content reached 0.6 μg/ml.When the ESC content reached 6 μg/ml,the colony growth was completely inhibited.It is indicated that visible light can inhibit the formation of conidia by producing red ESC from E.arachidis.4.Study on inoculation conditions and infection process of E.arachidisIn order to explore the infection process of E.arachidis,this paper clarified the optimal inoculation conditions suitable for E.arachidis.Light microscopy,scanning electron microscopy and transmission electron microscopy were used to systematically study the developmental process,infection process,host symptom changes and pathological changes of host cells in peanut leaf infected by E.arachidis.The results showed that the optimal temperature for inoculation of peanut leaves by conidia suspension was 25 °C,and the minimum duration of humidity was 4 h.The young peanut leaves were more susceptible to inoculation than the old leaves.The incidence of inoculation points of leaves 1 and 2 was more than 90%,while that of leaves 7 was 33.30%.The conidia can germinate to form one or more germ tubes,and elongate into hyphae to spread on the surface of the leaves,randomly branching,and the top of the branched germ tube produced structures similar to the appressorium and penetration peg.Mucus-like substances were found in the structure of penetration peg.It is speculated that the mucus may be a glucan,which is to make the conidia better adhere to the surface of the leaf and produce a substance to promote the infection of pathogens.3 days after inoculation,the phenomenon that the hyphae directly invade the surface of the leaves was observed.At the same time,the peanut mesophyll cells were destroyed,the wall separation occurred,the host resistance reaction was obvious,and the lesions began to appear at the peanut leaf inoculation point. |
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